DNA in the genome is compacted with histone proteins as nucleosomes, fundamental organizational units of chromosomes in the cell nucleus. Nucleosomes are arrayed in fibers of chromatin, associated with nonhistone regulators, architectural proteins and RNA in the cell nucleus. The architecture of chromatin has an important role in the regulation of gene expression in the life of a cell. This project aims to investigate the very large, 14-component, chromatin remodeling protein complex called SWR1 in the budding yeast model organism. SWR1 is evolutionarily conserved from yeast to humans, and functions as an enzyme to catalyze exchange of the minor histone variant H2A.Z for canonical histone H2A, the bulk histone species in cells. H2A.Z specifically marks nucleosomes next to nucleosome-deficient promoter and enhancer elements, modulating recruitment and activity of the RNA polymerase machinery. The dysfunction of SWR1 and H2A.Z are implicated in disease, including cancer. The molecular mechanism of the H2A.Z exchange reaction is not well understood. The proposed work will systematically extend biochemical studies of purified, SWR1 mutant complexes deficient for individual components or specific domains. We will use established biochemical assays and a high-throughput fluorescence resonance energy transfer assay that quantifies the rate of histone exchange. We will develop a single-molecule imaging platform to detect specific reaction intermediates and measure their lifetimes during histone exchange. Detection of biochemical defects in SWR1 mutants will inform the roles of the corresponding normal components in the H2A.Z exchange pathway. In collaboration with accomplished structural biologists working at moderate and high resolution, we plan to gain high-resolution structural information on the SWR1 enzyme complex, free and bound to substrates, to increase knowledge of the structural basis of histone exchange, and assist future rational drug design and drug screening.